Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics

ABSTRACT

A self-addressable, self-assembling microelectronic device is designed and fabricated to actively carry out and control multi-step and multiplex molecular biological reactions in microscopic formats. These reactions include nucleic acid hybridization, antibody/antigen reaction, diagnostics, and biopolymer synthesis. The device can be fabricated using both microlithographic and micro-machining techniques. The device can electronically control the transport and attachment of specific binding entities to specific micro-locations. The specific binding entities include molecular biological molecules such as nucleic acids and polypeptides. The device can subsequently control the transport and reaction of analytes or reactants at the addressed specific micro-locations. The device is able to concentrate analytes and reactants, remove non-specifically bound molecules, provide stringency control for DNA hybridization reactions, and improve the detection of analytes. The device can be electronically replicated.

RELATED INFORMATION

This application is a continuation of application Ser. No. 09/128,718,filed Aug. 4, 1998, now issued as U.S. Pat. No. 7,314,708, which is acontinuation of application Ser. No. 08/725,976, filed Oct. 4, 1996, nowissued as U.S. Pat. No. 5,929,208, which is a continuation ofapplication Ser. No. 08/146,504, filed Nov. 1, 1993, now issued as U.S.Pat. No. 5,605,662, the contents of each are expressly incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention pertains to the design, fabrication, and uses of aself-addressable, self-assembling microelectronic system which canactively carry out and control multi-step and multiplex reactions inmicroscopic formats.

In particular, these reactions include molecular biological reactions,such as nucleic acid hybridizations, antibody/antigen reactions,clinical diagnostics, and biopolymer synthesis.

BACKGROUND OF THE INVENTION

Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein, many of which form the basis ofclinical diagnostic assays. These techniques include nucleic acidhybridization analysis, restriction enzyme analysis, genetic sequenceanalysis, and separation and purification of nucleic acids and proteins(See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, MolecularCloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989).

Most molecular biology techniques involve carrying out numerousoperations (e.g., pipetting) on a large number of samples. They areoften complex and time consuming, and generally require a high degree ofaccuracy. Many a technique is limited in its application by a lack ofsensitivity, specificity, or reproducibility. For example, problems withsensitivity and specificity have so far limited the application ofnucleic acid hybridization.

Nucleic acid hybridization analysis generally involves the detection ofa very small numbers of specific target nucleic acids (DNA or RNA) withprobes among a large amount of non-target nucleic acids. In order tokeen high specificity, hybridization is normally carried out under themost stringent condition, achieved through a combination of temperature,salts, detergents, solvents, chaotropic agents, and denaturants.

Multiple sample nucleic acid hybridization analysis has been conductedon a variety of filter and solid support formats (see G. A. Beltz etal., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossmam, K.Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985).One format, the so-called “dot blot” hybridization, involves thenon-covalent attachment of target DNAs to a filter, which aresubsequently hybridized with a radioisotope labeled probe(s). “Dot blot”hybridization gained wide-spread use, and many versions were developed(see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridization—APractical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press,Washington D.C., Chapter 4, pp. 73-111, 1985). It has been developed formultiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, inEPA 0228075, Jul. 8, 1987) and for the detection of overlapping clonesand the construction of genomic maps (G. A. Evans, in U.S. Pat. No.5,219,726, Jun. 15, 1993).

Another format, the so-called “sandwich” hybridization, involvesattaching oligonucleotide probes covalently to a solid support and usingthem to capture and detect multiple nucleic acid targets. (M. Ranki etal., Gene, 21, pp. 77-85, 1983; A. M. Palva, T. M. Ranki, and H. E.Soderlund, in UK Patent Application GB 2156074A, Oct. 2, 1985; T. M.Ranki and H. E. Soderlund in U.S. Pat. No. 4,563,419, Jan. 7, 1986; A.D. B. Malcolm and J. A. Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y.Stabinsky, in U.S. Pat. No. 4,751,177, Jan. 14, 1988; T. H. Adams etal., in PCT WO 90/01564, Feb. 22, 1990; R. B. Wallace et al. 6 NucleicAcid Res. 11, p. 3543, 1979; and B. J. Connor et al., 80 Proc. Natl.Acad. Sci. USA pp. 278-282; 1983).

Using the current nucleic acid hybridization formats and stringencycontrol methods, it remains difficult to detect low copy number (i.e.,1-100,000) nucleic acid targets even with the most sensitive reportergroups (enzyme, fluorophores, radioisotopes, etc.) and associateddetection systems (fluorometers, luminometers, photon counters,scintillation counters, etc.).

This difficulty is caused by several underlying problems associated withdirect probe hybridization. The first and the most serious problemrelates to the stringency control of hybridization reactions.Hybridization reactions are usually carried out under the most stringentconditions in order to achieve the highest degree of specificity.Methods of stringency control involve primarily the optimization oftemperature, ionic strength, and denaturants in hybridization andsubsequent washing procedures. Unfortunately, the application of thesestringency conditions causes a significant decrease in the number ofhybridized probe/target complexes for detection.

The second problem relates to the high complexity of DNA in mostsamples, particularly in human genomic DNA samples. When a sample iscomposed of an enormous number of sequences which are closely related tothe specific target sequence, even the most unique probe sequence has alarge number of partial hybridizations with non-target sequences.

The third problem relates to the unfavorable hybridization dynamicsbetween a probe and its specific target. Even under the best conditions,most hybridization reactions are conducted with relatively lowconcentrations of probes and target molecules. In addition, a probeoften has to compete with the complementary strand for the targetnucleic acid.

The fourth problem for most present hybridization formats is the highlevel of non-specific background signal. This is caused by the affinityof DNA probes to almost any material.

These problems, either individually or in combination, lead to a loss ofsensitivity and/or specificity for nucleic acid hybridization in theabove described formats. This is unfortunate because the detection oflow copy number nucleic acid targets is necessary for most nucleicacid-based clinical diagnostic assays.

Because of the difficulty in detecting low copy number nucleic acidtargets, the research community relies heavily on the polymerase chainreaction (PCR) for the amplification of target nucleic acid sequences(see M. A. Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, 1990). The enormous number of targetnucleic acid sequences produced by the PCR reaction improves thesubsequent direct nucleic acid probe techniques, albeit at the cost of alengthy and cumbersome procedure.

A distinctive exception to the general difficulty in detecting low copynumber target nucleic acid with a direct probe is the in-situhybridization technique. This technique allows low copy number uniquenucleic acid sequences to be detected in individual cells. In thein-situ format, target nucleic acid is naturally confined to the area ofa cell (˜20-50 μm²) or a nucleus (˜10 μm²) at a relatively high localconcentration. Furthermore, the probe/target hybridization signal isconfined to a morphologically distinct area; this makes it easier todistinguish a positive signal from artificial or non-specific signalsthan hybridization on a solid support.

Mimicking the in-situ hybridization, new techniques are being developedfor carrying out multiple sample nucleic acid hybridization analysis onmicro-formatted multiplex or matrix devices (e.g., DNA chips) (see M.Barinaga, 253 Science, pp. 1489; 1991. W. Bains 10 Bio/Technology, pp.757-758, 1992). These methods usually attach specific DNA sequences tovery small specific areas of a solid support, such as micro-wells of aDNA chip. These hybridization formats are micro-scale versions of theconventional “dot blot” and “sandwich” hybridization systems.

The micro-formatted hybridization can be used to carry out “sequencingby hybridization” (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991;W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of allpossible n-nucleotide oligomers (n-mers) to identify n-mers in anunknown DNA sample, which are subsequently aligned by algorithm analysisto produce the DNA sequence (R. Drmanac and R. Crkvenjakov, YugoslavPatent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114,1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1991; andR. Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13,1993).

There are two formats for carrying out SBH. The first format involvescreating an array of all possible n-mers on a support, which is thenhybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

Southern, United Kingdom Patent Application GB 8810400, 1988; E. M.Southern et al., 13 Genomics 1008, 1992, proposed using the first formatto analyze or sequence DNA. Southern identified a known single pointmutation using PCR amplified genomic DNA. Southern also described amethod for synthesizing an array of oligonucleotides on a solid supportfor SBH. However, Southern did not address how to achieve optimalstringency condition for each oligonucleotide on an array.

Fodor et al., 364 Nature, pp. 555-556, 1993, used an array of 1,0248-mer oligonucleotides on a solid support to sequence DNA. In this case,the target DNA was a fluorescently labeled single-stranded 12-meroligonucleotide containing only nucleotides A and C. 1 μmol (˜6×10¹¹molecules) of the 12-mer target sequence was necessary for thehybridization with the 8-mer oligomers on the array. The results showedmany mismatches. Like Southern, Fodor et al., did not address theunderlying problems of direct probe hybridization, such as stringencycontrol for multiplex hybridizations. These problems, together with therequirement of a large quantity of the simple 12-mer target, indicatesevere limitations to this SBH format.

Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used thesecond format to sequence several short (116 bp) DNA sequences. TargetDNAs were attached to membrane supports (“dot blot” format). Each filterwas sequentially hybridized with 272 labeled 10-mer and 11-meroligonucleotides. A wide range of stringency condition was used toachieve specific hybridization for each n-mer probe; washing timesvaried from 5 minutes to overnight, and temperatures from 0° C. to 16°C. Most probes required 3 hours of washing at 16° C. The filters had tobe exposed for 2 to 18 hours in order to detect hybridization signals.The overall false positive hybridization rate was 5% in spite of thesimple target sequences, the reduced set of oligomer probes, and the useof the most stringent conditions available.

Fodor et al., 251 Science 767-773, 1991, used photolithographictechniques to synthesize oligonucleotides on a matrix. Pirrung et al.,in U.S. Pat. No. 5,143,854, Sep. 1, 1992, teach large scalephotolithographic solid phase synthesis of polypeptides in an arrayfashion on silicon substrates.

In another approach of matrix hybridization, Beattie et al., in The 1992San Diego Conference: Genetic Recognition, November, 1992, used amicrorobotic system to deposit micro-droplets containing specific DNAsequences into individual microfabricated sample wells on a glasssubstrate. The hybridization in each sample well is detected byinterrogating miniature electrode test fixtures, which surround eachindividual microwell with an alternating current (AC) electric field.

Regardless of the format, current micro-scale DNA hybridization and SBHapproaches do not overcome the underlying physical problems associatedwith direct probe hybridization reactions. They require very high levelsof relatively short single-stranded target sequences or PCR amplifiedDNA, and produce a high level of false positive hybridization signalseven under the most stringent conditions. In the case of multiplexformats using arrays of short oligonucleotide sequences, it is notpossible to optimize the stringency condition for each individualsequence with any conventional approach because the arrays or devicesused for these formats can not change or adjust the temperature, ionicstrength, or denaturants at an individual location, relative to otherlocations. Therefore, a common stringency condition must be used for allthe sequences on the device. This results in a large number ofnon-specific and partial hybridizations and severely limits theapplication of the device. The problem becomes more compounded as thenumber of different sequences on the array increases, and as the lengthof the sequences decreases. This is particularly troublesome for SBH,which requires a large number of short oligonucleotide probes.

Nucleic acids of different size, charge, or conformation are routinelyseparated by electrophoresis techniques which can distinguishhybridization species by their differential mobility in an electricfield. Pulse field electrophoresis uses an arrangement of multipleelectrodes around a medium (e.g., a gel) to separate very large DNAfragments which cannot be resolved by conventional gel electrophoresissystems (see R. Anand and E. M. Southern in Gel Electrophoresis ofNucleic Acids—A Practical Approach, 2 ed., D. Rickwood and B. D. HamesEds., IRL Press, New York, pp. 101-122, 1990).

Pace, U.S. Pat. No. 4,908,112, Mar. 13, 1990, teaches usingmicro-fabrication techniques to produce a capillary gel electrophoresissystem on a silicon substrate. Multiple electrodes are incorporated intothe system to move molecules through the separation medium within thedevice.

Soane and Soane, U.S. Pat. No. 5,126,022, Jun. 30, 1992, teach that anumber of electrodes can be used to control the linear movement ofcharged molecules in a mixture through a gel separation medium containedin a tube. Electrodes have to be installed within the tube to controlthe movement and position of molecules in the separation medium.

Washizu, M. and Kurosawa, O., 26 IEEE Transactions on IndustryApplications 6, pp. 1165-1172, 1990, used high-frequency alternatingcurrent (AC) fields to orient DNA molecules in electric field linesproduced between microfabricated electrodes. However, the use of directcurrent (DC) fields is prohibitive for their work. Washizu 25 Journal ofElectrostatics 109-123, 1990, describes the manipulation of cells andbiological molecules using dielectrophoresis. Cells can be fused andbiological molecules can be oriented along the electric fields linesproduced by AC voltages between the microelectrode structures. However,the dielectrophoresis process requires a very high frequency AC (1 MHz)voltage and a low conductivity medium. While these techniques can orientDNA molecules of different sizes along the AC field lines, they cannotdistinguish between hybridization complexes of the same size.

As is apparent from the preceding discussion, numerous attempts havebeen made to provide effective techniques to conduct multi-step,multiplex molecular biological reactions. However, for the reasonsstated above, these techniques have been proved deficient. Despite thelong-recognized need for effective technique, no satisfactory solutionhas been proposed previously.

SUMMARY OF THE INVENTION

The present invention relates to the design, fabrication, and uses of aself-addressable self-assembling microelectronic system and device whichcan actively carry out controlled multi-step and multiplex reactions inmicroscopic formats. These reactions include, but are not limited to,most molecular biological procedures, such as nucleic acidhybridization, antibody/antigen reaction, and related clinicaldiagnostics. In addition, the claimed device is able to carry outmulti-step combinational biopolymer synthesis, including, but notlimited to, the synthesis of different oligonucleotides or peptides atspecific micro-locations.

The claimed device is fabricated using both microlithographic andmicro-machining techniques. The device has a matrix of addressablemicroscopic locations on its surface; each individual micro-location isable to electronically control and direct the transport and attachmentof specific binding entities (e.g., nucleic acids, antibodies) toitself. All micro-locations can be addressed with their specific bindingentities. Using this device, the system can be self-assembled withminimal outside intervention.

The device is able to control and actively carry out a variety of assaysand reactions. Analytes or reactants can be transported by free fieldelectrophoresis to any specific micro-location where the analytes orreactants are effectively concentrated and reacted with the specificbinding entity at said micro-location. The sensitivity for detecting aspecific analyte or reactant is improved because of the concentratingeffect. Any un-bound analytes or reactants can be removed by reversingthe polarity of a micro-location. Thus, the device also improves thespecificity of assays and reactions.

The device provides independent stringency control for hybridizationreactions at specific micro-locations. Thus all the micro-locations onthe matrix can have different stringency conditions at the same time,allowing multiple hybridizations to be conducted at optimal conditions.

The device also facilitates the detection of hybridized complexes ateach micro-location by using an associated optical (fluorescent orspectrophotometric) imaging detector system or an integrated sensingcomponent.

In addition, the active nature of the device allows complex multi-stepreactions to be carried out with minimal outside physical manipulations.If desired, a master device addressed with specific binding entities canbe electronically replicated or copied to another base device.

Thus, the claimed device can carry out multi-step and multiplexreactions with complete and precise electronic control, preferably witha micro-processor. The rate, specificity, and sensitivity of multi-stepand multiplex reactions are greatly improved at specific micro-locationsof the claimed device.

The present invention overcomes the limitations of the arrays anddevices for multi-sample hybridizations described in the background ofthe invention. Previous methods and devices are functionally passiveregarding the actual hybridization process. While sophisticatedphotolithographic techniques were used to make an array, ormicroelectronic sensing elements were incorporated for detection;previous devices did not control or influence the actual hybridizationprocess. They are not designed to actively overcome any of theunderlying physical problems associated with hybridization reactions.

This invention may utilize micro-locations of any size or shapeconsistent with the objective of the invention. In the preferredembodiment of the invention, micro-locations in the sub-millimeter rangeare used.

By “specific binding entity” is generally meant a biological orsynthetic molecule that has specific affinity to another molecule,through covalent bonding or non-covalent bonding. Preferably, a specificbinding entity contains (either by nature or by modification) afunctional chemical group (primary amine, sulfhydryl, aldehyde, etc.), acommon sequence (nucleic acids), an epitope (antibodies), a hapten, or aligand, that allows it to covalently react or non-covalently bind to acommon functional group on the surface of a micro-location. Specificbinding entities include, but are not limited to: deoxyribonucleic acids(DNA), ribonucleic acids (RNA), synthetic oligonucleotides, antibodies,proteins, peptides, lectins, modified polysaccharides, syntheticcomposite macromolecules, functionalized nanostructures, syntheticpolymers, modified/blocked nucleotides/nucleosides, modified/blockedamino acids, fluorophores, chromophores, ligands, chelates and haptens.

By “stringency control” is meant the ability to discriminate specificand non-specific binding interactions.

Thus, in a first aspect, the present invention features a device with anarray of electronically self-addressable microscopic locations. Eachmicroscopic location contains an underlying working direct current (DC)micro-electrode supported by a substrate. The surface of eachmicro-location has a permeation layer for the free transport of smallcounter-ions, and an attachment layer for the covalent coupling ofspecific binding entities.

By “array” or “matrix” is meant an arrangement of locations on thedevice. The locations can be arranged in two dimensional arrays, threedimensional arrays, or other matrix formats. The number of locations canrange from several to at least hundreds of thousands.

In a second aspect, this invention features a method for transportingthe binding entity to any specific micro-location on the device. Whenactivated, a micro-location can affect the free field electrophoretictransport of any charged functionalized specific binding entity directlyto itself. Upon contacting the specific micro-location, thefunctionalized specific binding entity immediately becomes covalentlyattached to the attachment layer surface of that specificmicro-location. Other micro-locations can be simultaneously protected bymaintaining them at the opposite potential to the charged molecules. Theprocess can be rapidly repeated until all the micro-locations areaddressed with their specific binding entities.

By “charged functionalized specific binding entity” is meant a specificbinding entity that is chemically reactive (i.e., capable of covalentattachment to a location) and carrying a net change (either positive ornegative).

In a third aspect, this inventions features a method for concentratingand reacting analytes or reactants at any specific micro-location on thedevice. After the attachment of the specific binding entities, theunderlying microelectrode at each micro-location continues to functionin a direct current (DC) mode. This unique feature allows relativelydilute charged analytes or reactant molecules free in solution to berapidly transported, concentrated, and reacted in a serial or parallelmanner at any specific micro-locations which are maintained at theopposite charge to the analyte or reactant molecules. Specificmicro-locations can be protected or shielded by maintaining them at thesame charge as the analytes or reactants molecules. This ability toconcentrate dilute analyte or reactant molecules at selectedmicro-locations greatly accelerates the reaction rates at thesemicro-locations.

When the desired reaction is complete, the micro-electrode potential canbe reversed to remove non-specific analytes or unreacted molecules fromthe micro-locations.

Specific analytes or reaction products may be released from anymicro-location and transported to other locations for further analysis;or stored at other addressable locations; or removed completely from thesystem.

The subsequent analysis of the analytes at the specific micro-locationsis also greatly improved by the ability to repulse non-specific entitiesfrom these locations.

In a fourth aspect, this invention features a method for improvingstringency control of nucleic acid hybridization reactions, comprisingthe steps of:

-   -   rapidly concentrating dilute target DNA and/or probe DNA        sequences at specific micro-location(s) where hybridization is        to occur;    -   rapidly removing non-specifically bound target DNA sequences        from specific micro-location(s) where hybridization has        occurred;    -   rapidly removing competing complementary target DNA sequences        from specific micro-location(s) where hybridization has        occurred;    -   raising electric potential to remove partially hybridized DNA        sequences (more than one base mis-match);    -   adjusting electric potential to improve the resolution of single        mis-match hybridizations (e.g., to identify point mutations);    -   applying independent electric potential control to individual        hybridization events occurring in the same bulk solution; and    -   using electric potential control to improve hybridization of        un-amplified target DNA sequences to arrays of capture        oligonucleotide probes.

In a fifth aspect, this invention features a method for synthesizingbiopolymers at micro-locations.

In a sixth aspect, this invention features a method for replicating amaster device.

In a seventh aspect, this invention features methods for detecting andanalyzing reactions that have occurred at the addressed micro-locationsusing self-addressed microelectronic devices with associated optical,optoelectronic or electronic detection systems or self-addressedmicroelectronic devices with integrated optical, optoelectronic orelectronic detection systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the cross-section of three self-addressable micro-locationsfabricated using microlithographic techniques.

FIG. 2 is the cross-section of a microlithographically fabricatedmicro-location.

FIG. 3 is a schematic representation of a self-addressable 64micro-location chip which was actually fabricated, addressed witholigonucleotides, and tested.

FIG. 4 shows particular attachment chemistry procedure which allowsrapid covalent coupling of specific oligonucleotides to the attachmentsurface of a micro-location.

FIG. 5 is a blown-up schematic diagram of a micromachined 96micro-locations device.

FIG. 6 is the cross-section of a micro-machined device.

FIG. 7 shows the mechanism the device uses to electronically concentrateanalyte or reactant molecules

FIG. 8 shows the self-directed assembly of a device with three specificoligonucleotide binding entities (SSO-A, SSO-B, and SSO-C).

FIG. 9 shows an electronically controlled hybridization process withsample/target DNA being concentrated at micro-locations containingspecific DNA capture sequences.

FIG. 10 shows an electronically directed serial hybridization process.

FIG. 11 shows the electronic stringency control (ESC) of a hybridizationprocess for determining single point mutations.

FIG. 12 shows a scheme for the detection of hybridized DNA without usinglabeled DNA probe, i.e., electronically controlled fluorescent dyedetection process.

FIG. 13 shows a scheme of electronically controlled replication ofdevices.

FIG. 14 shows a scheme of electronically directed combinatorialsynthesis of oligonucleotides.

DETAILED DESCRIPTION OF THE INVENTION

The devices and the related methodologies of this invention allowimportant molecular biology and diagnostic reactions to be carried outunder complete electronic control. The basic concept of this inventionis a microelectronic device with specially designed addressablemicroscopic locations. Each micro-location has a derivatized surface forthe covalent attachment of specific binding entities (i.e., anattachment layer), a permeation layer, and an underlying direct current(DC) microelectrode. After the initial fabrication of the basicmicroelectronic structure, the device is able to self-direct theaddressing of each specific micro-location with specific bindingentities. The self-addressed device is combinatorial, and multiplexreactions at any of its micro-locations. The device is able toelectronically direct and control the rapid movement and concentrationof analytes and reactants to or from any of its micro-locations. Theability of the device to electronically control the dynamic aspects ofvarious reactions provides a number of new and important advantages andimprovements.

The concepts and embodiments of this invention are described in threesections. The first section, “Design and Fabrication of the BasicDevices,” describes the design of the basic underlying microelectronicdevice and the fabrication of the device using microlithographic andmicromachining techniques. The second section, “Self-Directed Addressingof the Devices,” describes the self-addressing and self-assembly of thedevice, specifically the rapid transport and attachment of specificbinding entities to each micro-location. The third section,“Applications of the Devices,” describes how the device provideselectronic control of various multi-step, combinatorial, and multiplexreactions. This section also describes the various uses and applicationsof the device.

(1) Design and Fabrication of the Basic Devices

In order for a device to carry out multi-step and multiplex reactions,its crucial electronic components must be able to maintain activeoperation in aqueous solutions. To satisfy this requirement, eachmicro-location must have an underlying functioning DC modemicro-electrode. Other considerations for the design and fabrication ofa device include, but are not limited to, materials compatibilities,nature of the specific binding entities and the subsequent reactants andanalytes, and the number of micro-locations.

By “a functioning DC mode micro-electrode” is meant a micro-electrodebiased either positively or negatively, operating in a direct currentmode (either continuous or pulse), which can affect or cause the freefield electrophoretic transport of charged specific binding entities,reactants, or analytes to or from any location on the device, or in thesample solution.

Within the scope of this invention, the free field electrophoretictransport of molecules is not dependent on the electric field producedbeing bounded or confined by dielectrical material.

A device can be designed to have as few as two addressablemicro-locations or as many as hundreds of thousands of micro-locations.In general, a complex device with a large number of micro-locations isfabricated using microlithography techniques. Fabrication is carried outon silicon or other suitable substrate materials, such as glass, silicondioxide, plastic, or ceramic materials. These microelectronic “chip”designs would be considered large scale array or multiplex analysisdevices. A device with a small number of micro-locations would befabricated using micro-machining techniques.

Addressable micro-locations can be of any shape, preferably round,square, or rectangular. The size of an addressable micro-location can beof any size, preferably range from sub-micron (˜0.5 μm) to severalcentimeters (cm), with 5 μm to 100 μm being the most preferred sizerange for devices fabricated using microlithographic techniques, and 100μm to 5 millimeters being the most preferred size range for devicesfabricated using the micro-machining techniques. To make micro-locationssmaller than the resolution of microlithographic methods would requiretechniques such as electron beam lithography, ion beam lithography, ormolecular beam epitaxy. While microscopic locations are desirable foranalytical and diagnostic type applications, larger addressablelocations (e.g., larger than 2 mm) are desirable for preparative scalebiopolymer synthesis.

After micro-locations have been created by using microlithographicand/or micro-machining techniques, chemical techniques are used tocreate the specialized attachment and permeation layers which wouldallow the DC mode micro-electrodes under the micro-locations to: (1)affect or cause the free field electrophoretic transport of specific(charged) binding entities from any location; (2) concentrate andcovalently attach the specific binding entities to the speciallymodified surface of the specific micro-location; and (3) continue toactively function in the DC mode after the attachment of specificbinding entities so that other reactants and analytes can be transportedto or from the micro-locations.

Design Parameters (Microlithography)

FIG. 1 shows a basic design of self-addressable micro-locationsfabricated using microlithographic techniques. The three micro-locations(10) (ML-1, ML-2, ML-3) are formed on the surface of metal sites (12)which have been deposited on an insulator layer/base material. The metalsites (12) serve as the underlying microelectrode structures (10). Aninsulator material separates the metal sites (12) from each other.Insulator materials include, but are not limited to, silicon dioxide,glass, resist, rubber, plastic, or ceramic materials.

FIG. 2 shows the basic features of an individual micro-location (10)formed on a microlithographically produced metal site (12). Theaddressable micro-location is formed on the metal site (12), andincorporates an oxidation layer (20), a permeation layer (22), anattachment layer (24), and a binding entity layer (26). The metal oxidelayer provides a base for the covalent coupling of the permeation layer.The permeation layer provides spacing between the metal surface and theattachment/binding entity layers and allows solvent molecules, smallcounter-ions, and gases to freely pass to and from the metal surface.The thickness of the permeation layer for microlithographically produceddevices can range from approximately 1 nanometers (nm) to 10 microns(μm), with 2 nm to 1 μm being the most preferred. The attachment layerprovides a base for the covalent binding of the binding entities. Thethickness of the attachment layer for microlithographically produceddevices can range from 0.5 nm to 1 μm, with 1 nm to 200 nm being themost preferred. In some cases, the permeation and attachment layers canbe formed from the same material. The specific binding entities arecovalently coupled to the attachment layer, and form the specificbinding entity layer. The specific binding entity layer is usually amono-layer of the specific binding molecules. However, in some cases thebinding entity layer can have several or even many layers of bindingmolecules.

Certain design and functional aspects of the permeation and attachmentlayer are dictated by the physical (e.g., size and shape) and thechemical properties of the specific binding entity molecules. They arealso dictated to some extent by the physical and chemical properties ofthe reactant and analyte molecules, which will be subsequentlytransported and bound to the micro-location. For example,oligonucleotide binding entities can be attached to one type ofmicro-location surface without causing a loss of the DC mode function,i.e., the underlying micro-electrode can still cause the rapid freefield electrophoretic transport of other analyte molecules to or fromthe surface to which the oligonucleotide binding entities are attached.However, if large globular protein binding entities (e.g., antibodies)are attached to the same type of surface, they may effectively insulatethe surface and cause a decrease or a complete loss of the DC modefunction. Appropriate modification of the attachment layer would have tobe carried out so as to either reduce the number of large bindingentities (e.g., large globular proteins) or provide spacing between thebinding entities on the surface.

The spacing between micro-locations is determined by the ease offabrication, the requirement for detector resolution betweenmicro-locations, and the number of micro-locations desired on a device.However, particular spacings between micro-locations, or specialarrangement or geometry of the micro-locations is not necessary fordevice function, in that any combination of micro-locations (i.e.,underlying micro-electrodes) can operate over the complete device area.Nor is it necessary to enclose the device or confine the micro-locationswith dielectric boundaries. This is because complex electronic fieldpatterns or dielectric boundaries are not required to selectively move,separate, hold, or orient specific molecules in the space or mediumbetween any of the electrodes. The device accomplishes this by attachingthe specific binding molecules and subsequent analytes and reactants tothe surface of an addressable micro-location. Free field electrophoreticpropulsion provides for the rapid and direct transport of any chargedmolecule between any and all locations on the device.

As the number of micro-locations increases beyond several hundred, thecomplexity of the underlying circuitry of the micro-locations increases.In this case the micro-location grouping patterns have to be changed andspacing distances increased proportionally, or multi-layer circuitry canbe fabricated into the basic device.

In addition to micro-locations which have been addressed with specificbinding entities, a device will contain some un-addressed, or plainmicro-locations which serve other functions. These micro-locations canbe used to store reagents, to temporarily hold reactants or analytes,and as disposal units for excess reactants, analytes, or otherinterfering components in samples. Other un-addressed micro-locationscan be used in combination with the addressed micro-locations to affector influence the reactions that are occurring at these specificmicro-locations. These micro-locations add to intra-device activity andcontrol. It is also possible for the micro-locations to interact andtransport molecules between two separate devices. This provides amechanism for loading a working device with binding entities orreactants from a storage device, and for copying or replicating adevice.

FIG. 3 shows a matrix type device containing 64 addressablemicro-locations (30). A 64 micro-location device is a convenient design,which fits with standard microelectronic chip packaging components. Sucha device is fabricated on a silicon chip substrate approximately 1.5cm×1.5 cm, with a central area approximately 750 μm×750 μm containingthe 64 micro-locations. Each micro-location (32) is approximately 50 μmsquare with 50 μm spacing between neighboring micro-locations.Connective circuitry for each individual underlying micro-electrode runsto an outside perimeter (10 mm×10 mm) of metal contact pads (300 μmsquare) (34). A raised inner perimeter can be formed between the areawith the micro-locations and the contact pads, producing a cavity whichcan hold approximately 2 to 10 microliters (μl) of a sample solution.The “chip” can be mounted in a standard quad package, and the chipcontact pads (34) wired to the quad package pins. The packaged chip canthen be plugged into a microprocessor controlled DC power supply andmultimeter apparatus which can control and operate the device.

Fabrication Procedures (Microlithography)

Microlithography Fabrication Steps

General microlithographic or photolithographic techniques can be usedfor the fabrication of the complex “chip” tape device which has a largenumber of small micro-locations. While the fabrication of devices doesnot require complex photolithography, the selection of materials and therequirement that an electronic device function actively in aqueoussolutions does require special considerations.

The 64 micro-location device (30) shown in FIG. 3 can be fabricatedusing relatively simple mask design and standard microlithographictechniques. Generally, the base substrate material would be a 1 to 2centimeter square silicon wafer or a chip approximately 0.5 millimeterin thickness. The silicon chip is first overcoated with a 1 to 2 μmthick silicon dioxide (SiO₂) insulation coat, which is applied by plasmaenhanced chemical vapor deposition (PECVD).

In the next step, a 0.2 to 0.5 μm metal layer (e.g., aluminum) isdeposited by vacuum evaporation. In addition to aluminum, suitablemetals for circuitry include gold, silver, tin, copper, platinum,palladium, carbon, and various metal combinations. Special techniquesfor ensuring proper adhesion to the insulating substrate materials(SiO₂) are used with different metals.

The chip is next overcoated with a positive photoresist (Shipley,Microposit AZ 1350 J), masked (light field) with the circuitry pattern,exposed and developed. The photosolubilized resist is removed, and theexposed aluminum is etched away. The resist island is now removed,leaving the aluminum circuitry pattern on the chip. This includes anoutside perimeter of metal contact pads, the connective circuitry(wires), and the center array of micro-electrodes which serve as theunderlying base for the addressable micro-locations.

Using PECVD, the chip is overcoated first with a 0.2 to 0.4 micron layerof SiO₂, and then with a 0.1 to 0.2 micron layer of silicon nitride(Si₃N₄). The chip is then covered with positive photoresist, masked forthe contact pads and micro-electrode locations, exposed, and developed.Photosolubilized resist is removed, and the SiO₂ and Si₃N₄ layers areetched away to expose the aluminum contact pads and micro-electrodes.The surrounding island resist is then removed, the connective wiringbetween the contact pads and the micro-electrodes remains insulated bythe SiO₂ and Si₃N₄ layers.

The SiO₂ and Si₃N₄ layers provide important properties for thefunctioning of the device. First, the second SiO₂ layer has bettercontact and improved sealing with the aluminum circuitry. It is alsopossible to use resist materials to insulate and seal. This preventsundermining of the circuitry due to electrolysis effects when themicro-electrodes are operating. The final surface layer coating of Si₃N₄is used because it has much less reactivity with the subsequent reagentsused to modify the micro-electrode surfaces for the attachment ofspecific binding entities.

Permeation and Attachment Layer Formation Steps

At this point the micro-electrode locations on the device are ready tobe modified with a specialized permeation and attachment layer. Thisrepresents the most important aspect of the invention, and is crucialfor the active functioning of the device. The objective is to create onthe micro-electrode an intermediate permeation layer with selectivediffusion properties and an attachment surface layer with optimalbinding properties. The attachment layer should have from 10⁵ to 10⁷functionalized locations per square micron (μm²) for the optimalattachment of specific binding entities. However, the attachment ofspecific binding entities must not overcoat or insulate the surface soas to prevent the underlying micro-electrode from functioning. Afunctional device requires some fraction (˜5% to 25%) of the actualmetal micro-electrode surface to remain accessible to solvent (H₂O)molecules, and to allow the diffusion of counter-ions (e.g., Na⁺ andCl⁻) and electrolysis gases (e.g., O₂ and H₂) to occur.

The intermediate permeation layer must also allow diffusion to occur.Additionally, the permeation layer should have a pore limit propertywhich inhibits or impedes the larger binding entities, reactants, andanalytes from physical contact with the micro-electrode surface. Thepermeation layer keeps the active microelectrode surface physicallydistinct from the binding entity layer of the micro-location.

In terms of the primary device function, this design allows theelectrolysis reactions required for electrophoretic transport to occuron micro-electrode surface, but avoids adverse electrochemical effectsto the binding entities, reactants, and analytes.

One preferred procedure for the derivatization of the metalmicro-electrode surface uses aminopropyltriethoxy silane (APS). APSreacts readily with the oxide and/or hydroxyl groups on metal andsilicon surfaces. APS provides a combined permeation layer andattachment layer, with primary amine groups for the subsequent covalentcoupling of binding entities. In terms of surface binding sites, APSproduces a relatively high level of functionalization (i.e., a largenumber of primary amine groups) on slightly oxidized aluminum surfaces,an intermediate level of functionalization on SiO₂ surfaces, and verylimited functionalization of Si₃N₄ surfaces.

The APS reaction is carried out by treating the whole device (e.g., achip) surface for 30 minutes with a 10% solution of APS in toluene at50° C. The chip is then washed in toluene, ethanol, and then dried forone hour at 50° C. The micro-electrode metal surface is functionalizedwith a large number of primary amine groups (10⁵ to 10⁶ per squaremicron). Binding entities can now be covalently bound to the derivatizedmicro-electrode surface.

The APS procedure works well for the attachment of oligonucleotidebinding entities. FIG. 4 shows the mechanism for the attachment of3′-terminal aldehyde derivatized oligonucleotides (40) to an APSfunctionalized surface (42). While this represents one of the preferredapproaches, a variety of other attachment reactions are possible forboth the covalent and non-covalent attachment of many types of bindingentities.

Design and Fabrication (Micro-Machining)

This section describes how to use micro-machining techniques (e.g.,drilling, milling, etc.) or non-lithographic techniques to fabricatedevices. In general, these devices have relatively largermicro-locations (>100 microns) than those produced by microlithography.These devices could be used for analytical applications, as well as forpreparative type applications, such as biopolymer synthesis. Largeaddressable locations could be fabricated in three dimensional formats(e.g., tubes or cylinders) in order to carry a large amount of bindingentities. Such devices could be fabricated using a variety of materials,including, but not limited to, plastic, rubber, silicon, glass (e.g.,microchannelled, microcapillary, etc.), or ceramics. In the case ofmicromachined devices, connective circuitry and larger electrodestructures can be printed onto materials using standard circuit boardprinting techniques known to those skilled in the art.

Addressable micro-location devices can be fabricated relatively easilyusing micro-machining techniques. FIG. 5 is a schematic of arepresentative 96 micro-location device. This micro-location device isfabricated from a suitable material stock (2 cm×4 cm×1 cm), by drilling96 proportionately spaced holes (1 mm in diameter) through the material.An electrode circuit board (52) is formed on a thin sheet of plasticmaterial stock, which fit precisely over the top of the micro-locationcomponent (54). The underside of the circuit board contains theindividual wires (printed circuit) to each micro-location (55). Shortplatinum electrode structures (˜3-4 mm) (62) are designed to extendeddown into the individual micro-location chambers (57). The printedcircuit wiring is coated with a suitable water-proof insulatingmaterial. The printed circuit wiring converges to a socket, which allowsconnection to a multiplex switch controller (56) and DC power supply(58). The device is partially immersed and operates in a common bufferreservoir (59).

While the primary function of the micro-locations in devices fabricatedby micro-machining and microlithography techniques is the same, theirdesigns are different. In devices fabricated by microlithography, thepermeation and attachment layers are formed directly on the underlyingmetal micro-electrode. In devices fabricated by micro-machiningtechniques, the permeation and attachment layers are physicallyseparated from their individual metal electrode structure (62) by abuffer solution in the individual chamber or reservoir (57) (see FIG.6). In micro-machined devices the permeation and attachment layers canbe formed using functionalized hydrophilic gels, membranes, or othersuitable porous materials.

In general, the thickness of the combined permeation and attachmentlayers ranges from 10 μm to 10 mm. For example, a modified hydrophilicgel of 26% to 35% polyacrylamide (with 0.1% polylysine), can be used topartially fill (˜0.5 mm) each of the individual micro-location chambersin the device. This concentration of gel forms an ideal permeation layerwith a pore limit of from 2 nm to 3 nm. The polylysine incorporated intothe gel provides primary amine functional groups for the subsequentattachment of specific binding entities. This type of gel permeationlayer allows the electrodes to function actively in the DC mode When theelectrode is activated the gel permeation layer allows smallcounter-ions to pass through it, but the larger specific binding entitymolecules are concentrated on the outer surface. Here they becomecovalently bonded to the outer layer of primary amines, whicheffectively becomes the attachment layer.

An alternative technique for the formation of the permeation andattachment layers is to incorporate into the base of each micro-locationchamber a porous membrane material. The outer surface of the membrane isthen derivatized with chemical functional groups to form the attachmentlayer. Appropriate techniques and materials for carrying out thisapproach are known to those skilled in the art.

The above description for the design and fabrication of a device shouldnot be considered as a limit to other variations or forms of the basicdevice. Many variations of the device with larger or smaller numbers ofaddressable micro-locations are envisioned for different analytical andpreparative applications. Variations of the device with largeraddressable locations are envisioned for preparative biopolymersynthesis applications. Variations are also contemplated aselectronically addressable and controllable reagent dispensers for usewith other devices, including those produced by microlithographictechniques.

(2) Self-Directed Addressing of the Devices

The claimed devices are able to electronically self-address eachmicro-location with a specific binding entity. The device itselfdirectly affects or causes the transport and attachment of specificbinding entities to specific micro-locations. The device self-assemblesitself in the sense that no outside process, mechanism, or equipment isneeded to physically direct, position, or place a specific bindingentity at a specific micro-location. This self-addressing process isboth rapid and specific, and can be carried out in either a serial orparallel manner.

A device can be serially addressed with specific binding entities bymaintaining the selected micro-location in a DC mode and at the oppositecharge (potential) to that of a specific binding entity. All othermicro-locations are maintained at the same charge as the specificbinding entity. In cases where the binding entity is not in excess ofthe attachment sites on the micro-location, it is necessary to activateonly one other micro-electrode to affect the electrophoretic transportto the specific micro-location. The specific binding entity is rapidlytransported (in a few seconds, or preferably less than a second) throughthe solution, and concentrated directly at the specific micro-locationwhere it immediately becomes covalently bonded to the special surface.The ability to electronically concentrate reactants or analytes (70) ona specific micro-location (72) is shown in FIG. 7. All othermicro-locations remain unaffected by that specific binding entity. Anyunreacted binding entity is removed by reversing the polarity of thatspecific micro-location, and electrophoresing it to a disposal location.The cycle is repeated until all desired micro-locations are addressedwith their specific binding entities. FIG. 8 shows the serial processfor addressing specific micro-locations (81, 83, 85) with specificoligonucleotide binding entities (82, 84, 86).

The parallel process for addressing micro-locations simply involvessimultaneously activating a large number (particular group or line) ofmicro-electrodes so that the same specific binding entity istransported, concentrated, and reacted with more than one specificmicro-locations.

(3) Applications of the Devices

Once a device has been self-addressed with specific binding entities, avariety of molecular biology type multi-step and multiplex reactions andanalyses can be carried out on the device. The devices of this inventionare able to electronically provide active or dynamic control over anumber of important reaction parameters. This electronic control leadsto significant improvements in reaction rates, specificities, andsensitivities. The improvements in these reaction parameters come fromthe ability of the device to electronically control and affect: (1) therapid transport of reactants or analytes to a specific micro-locationcontaining attached specific binding entities; (2) improvement inreaction rates due to the concentrated reactants or analytes reactingwith the specific binding entities at that specific micro-location; and(3) the rapid and selective removal of un-reacted and non-specificallybound components from that micro-location. These advantages are utilizedin a novel process called “electronic stringency control”.

The self-addressed devices of this invention are able to rapidly carryout a variety of micro-formatted multi-step and/or multiplex reactionsand procedures; which include, but are not limited to:

-   -   DNA and RNA hybridizations procedures and analysis in        conventional formats, and new improved matrix formats;    -   molecular biology reaction procedures, e.g., restriction enzyme        reactions and analysis, ligase reactions, kinasing reactions,        and amplification procedures;    -   antibody/antigen reaction procedures involving large or small        antigens and haptens;    -   diagnostic assays, e.g., hybridization analysis, gene analysis,        fingerprinting, and immunodiagnostics;    -   biomolecular conjugation procedures (i.e. the covalent and        non-covalent labeling of nucleic acids, enzymes, proteins, or        antibodies with reporter groups);    -   biopolymer synthesis procedures, e.g., combinatorial synthesis        of oligonucleotides or peptides;    -   water soluble synthetic polymer synthesis, e.g., carbohydrates        or linear polyacrylates; and    -   macromolecular and nanostructure (nanometer size particles and        structures) synthesis and fabrication.        Nucleic Acid Hybridization

Nucleic acid hybridizations are used as examples of this inventionbecause they characterize the most difficult multi-step and multiplexreactions.

The claimed device and methods allow nucleic acid hybridization to becarried out in a variety of conventional and new formats. The ability ofthe device to electronically control reaction parameters greatlyimproves nucleic acid hybridization analysis, particularly the abilityof the device to provide electronic stringency control (ESC).

By “nucleic acid hybridization” is meant hybridization between allnatural and synthetic forms and derivatives of nucleic acids, including:deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polynucleotides andoligonucleotides.

Conventional hybridization formats, such as “dot blot” hybridization and“sandwich” hybridization, can be carried out with the claimed device aswell as large scale array or matrix formats.

As an example, a device for DNA hybridization analysis is designed,fabricated, and used in the following manner. Arrays of micro-locationsare first fabricated using microlithographic techniques. The number ofaddressable micro-locations on an array depends on the final use. Thedevice is rapidly self-addressed in a serial manner with a group ofspecific oligonucleotides. In this case, the specific oligonucleotidesare 3′-terminal aldehyde functionalized oligonucleotides (in the rangeof 6-mer to 100-mer). The aldehyde functional group allows for covalentattachment to the specific micro-location attachment surface (see FIG.4). This group of specific oligonucleotides can be readily synthesizedon a conventional DNA synthesizer using conventional techniques.

The synthesis of each specific oligonucleotide is initiated from aribonucleotide controlled pore glass (CPG) support. Thus, the3′-terminal position contains a ribonucleotide, which is then easilyconverted after synthesis and purification to a terminal dialdehydederivative by periodate oxidation. The aldehyde containingoligonucleotides (40) will react readily with the primary aminefunctional groups on the surface of micro-locations by a Schiff's basereaction process.

The electronic addressing of the device with specific oligonucleotidesis shown in FIG. 8. The addressing of the first specific micro-location(ML-1) (81) with its specific sequence oligonucleotide (SSO-1) (82) isaccomplished by maintaining the specific microelectrode (ML-1) at apositive DC potential, while all other microelectrodes are maintained ata negative potential (FIG. 8(A)). The aldehyde functionalized specificsequence (SSO-1) in aqueous buffered solution is free fieldelectrophoresed to the ML-1 address, where it concentrates (>10⁶ fold)and immediately becomes covalently bound to the surface of ML-1 (81).All other microelectrodes are maintained negative, and remain protectedor shielded from reacting with SSO-1 sequence (82). The ML-1 potentialis then reversed to negative (−) to electrophores any unreacted SSO-1 toa disposal system. The cycle is repeated, SSO-2 (84) --->ML-2 (83),SSO-3 (86) --->ML-3 (85), SSO-n --->ML-n until all the desiredmicro-locations are addressed with their specific DNA sequences (FIG.8(D)).

Another method for addressing the device is to transport specificbinding entities such as specific oligonucleotides from an electronicreagent supply device. This supply device would hold a large quantity ofbinding entities or reagents and would be used to load analyticaldevices. Binding entities would be electronically trans-ported betweenthe two devices. Such a process eliminates the need for physicalmanipulations, such as pipetting, in addressing a device with bindingentities.

Yet another method for addressing the device is to carry out thecombinatorial synthesis of the specific oligonucleotides at the specificmicro-locations. Combinatorial synthesis is described in a latersection.

After the device is addressed with specific DNA sequences, themicro-locations on the array device remain as independent working directcurrent (DC) electrodes. This is possible because the attachment to theelectrode surface is carried out in such a manner that the underlyingmicro-electrode does not become chemically or physically insulated. Eachmicro-electrode can still produce the strong direct currents necessaryfor the free field electrophoretic transport of other charged DNAmolecules to and from the micro-location surface. The DNA array deviceprovides complete electronic control over all aspects of the DNAhybridization and any other subsequent reactions.

An example of an electronically controlled hybridization process isshown in FIG. 9. In this case, each addressable micro-location has aspecific capture sequence (90). A sample solution containing target DNA(92) is applied to the device. All the micro-locations are activated andthe sample DNA is concentrated at the micro-locations (FIG. 9(B)).Target DNA molecules from the dilute solution become highly concentratedat the micro-locations, allowing very rapid hybridization to thespecific complementary DNA sequences on the surface. Reversal of themicro-electrode potential repels all unhybridized DNA from themicro-locations, while the target DNA remains hybridized (FIG. 9(C)). Insimilar fashion, reporter probes are hybridized in subsequent steps todetect hybridized complexes.

The electronic control of the hybridization process significantlyimproves the subsequent detection of the target DNA molecules byenhancing the overall hybridization efficiency and by removingnon-specific DNA from the micro-location areas. It is expected that10,000 to 100,000 copies of target sequences in un-amplified genomic DNAwill be detectable. Hybridization reactions of this type can be carriedout in a matter of minutes, with minimal outside manipulations.Extensive washing is not necessary.

Another common format for DNA hybridization assays involves havingtarget DNAs immobilized on a surface, and then hybridizing specificprobes to these target DNAs. This format can involve either the sametarget DNAs at multiple locations, or different target DNAs at specificlocations. FIG. 10 shows an improved version of this serialhybridization format. In this case micro-locations (101-107) areaddressed with different capture DNAs. These are hybridized in a serialfashion with different sequence specific oligonucleotides (108,109). Themicro-locations are sequentially biased positive to transport moleculesto itself and then biased negative to transport molecules to the nextmicro-location. Specifically hybridized DNA will remain at themicro-location regardless of electrode potential. The sequence specificoligonucleotides can be labeled with a suitable reporter group such as afluorophore.

The claimed device is able to provide electronic stringency control.Stringency control is necessary for hybridization specificity, and isparticularly important for resolving one base mismatches in pointmutations. FIG. 11 shows how electronic stringency control can be usedfor improving hybridization specificity for one base mismatch analysis.The electronic stringency control can also be applied to multiple-basemismatch analysis.

Perfectly matched DNA hybrids (110) are more stable than mismatched DNA(112) hybrids. By biasing the micro-locations negative (FIG. 11(B)) anddelivering a defined amount of power in a given time, it is possible todenature or remove the mismatched DNA hybrids while retaining theperfectly matched DNA hybrids (FIG. 11(C)). In a further refinement, theclaimed device provides independent stringency control to each specifichybridization reaction occurring on the device. With a conventional orpassive array format, it is impossible to achieve optimal stringency forall the hybridization events which are occurring in the samehybridization solution. However, the active array devices of thisinvention are able to provide different electronic stringency tohybridizations at different micro-locations, even though they areoccurring in the same bulk hybridization solution. This attributeovercomes a major limitation to conventional matrix hybridizationformats, sequencing by hybridization (SBH) formats, and other multiplexanalyses.

The ability to provide electronic stringency control to hybridizationsalso provides mechanisms for detecting DNA hybridization withoutreporter group labeled DNA probe. It provides a way to carry out a moredirect detection of the hybridization process itself. A fluorescent dyedetection process is shown in FIG. 12 and described in Examples 4 and 6.Direct detection of DNA hybrids can be achieved by using DNA bindingdyes such as ethidium bromide. The dye binds to both double-stranded andsingle-stranded DNA but with a greater affinity for the former. In FIG.12(B) positively charged dye (122) is transported to negatively biasedmicro-locations. The dye binds to both hybridized (120) and unhybridized(121) DNA sequences (FIG. 12 c). By biasing the micro-locations positiveand delivering a defined amount of power in a given amount of time, thedye molecules bound to unhybridized micro-locations is selectivelyremoved. The amount of power applied does not adversely affect the DNAhybrids.

The hybridized DNAs with associated dye molecules are then fluorescentlydetected using associated or integrated optical systems.

The following reiterates the important advantages the devices of thisinvention provide for nucleic acid hybridization reactions and analysis:

-   -   (1) The rapid transport of dilute target DNA and/or probe DNA        sequences to specific microlocation(s) where hybridization is to        occur. This process takes place in no more than a few seconds.    -   (2) Concentrating dilute target DNA and/or probe DNA sequences        at specific micro-location(s) where hybridization is to occur.        The concentrating effect can be well over a million fold (>10⁶).    -   (3) The rapid removal of non-specifically bound target DNA        sequences from specific microlocation(s) where hybridization has        occurred. This process takes 10 to 20 seconds.    -   (4) Rapid removal of competing complementary target DNA        sequences from specific micro-location(s) where hybridization        has occurred. This process takes 10 to 20 seconds.    -   (6) The ability to carry out a complete hybridization process in        several minutes.    -   (7) The ability to carry out a hybridization process with        minimal outside manipulations or washing steps.    -   (8) The use of electronic stringency control (ESC) to remove        partially hybridized DNA sequences.    -   (9) The ability to carry out hybridization analysis of        un-amplified genomic target DNA sequences in the 1000 to 100,000        copy range.    -   (10) The use of ESC to improve the resolution of single base        mis-match hybridizations (point mutations).    -   (11) The use of ESC to provide individual stringency control in        matrix hybridizations.    -   (12) Improving the detection of hybridization event by removing        non-specific background components.    -   (13) The development of new procedures which eliminate the need        for using covalently labeled reporter probes or target DNA to        detect the hybridization events.        Reproduction of Devices

In addition to separately addressing individual devices with specificbinding entities, it is also possible to produce a master device, whichcan copy specific binding entities to other devices. This representsanother method for the production of devices. The process for thereplication of devices is shown in FIG. 13. A master device containingmicro-locations which have been addressed with specific bindingsequences is hybridized with respective complementary DNA sequences(130). These complementary sequences are activated and thus capable ofcovalent binding to the micro-location attachment layer.

An unaddressed sister device (132) containing an attachment layer isaligned with the hybridized master device (FIG. 13(B)). The masterdevice micro-locations are biased negative and the sister devicemicro-locations are biased positive. The DNA hybrids are denatured andare transported to the sister device, where the activated DNA sequencebinds covalently to the micro-location (FIG. 13(C)). The process can beperformed in parallel or in series, depending on the device geometry sothat crosstalk between the micro-locations is minimized. The hybrids canbe denatured by applying a sufficient negative potential or by using apositively charged chaotropic agent or denaturant.

Detection System

In the case of fluorescent binding reactions, it is possible to use anepifluorescent type microscopic detection system for the analysis of thebinding reactions. The sensitivity of the system depends on theassociated imaging detector element (CCD, ICCD, Microchannel Plate) orphoton counting PMT system. One alternative is to associate a sensitiveCCD detector or avalanche photodiode (APD) detector directly with thedevice in a sandwich arrangement. Another alternative is to integrateoptoelectronic or microelectronics detection in the device.

Combinatorial Biopolymer Synthesis

The devices of this invention are also capable of carrying outcombinatorial synthesis of biopolymers such as oligonucleotides andpeptides. Such a process allows self-directed synthesis to occur withoutthe need for any outside direction or influence. This combinatorialsynthesis allows very large numbers of sequences to be synthesized on adevice. The basic concept for combinatorial synthesis involves the useof the device to transport, concentrate, and react monomers, couplingreagents, or deblocking reagents at the addressable micro-locations. Theconcept capitalizes on the ability of the device to protect certainlocations from the effects of nearby reagents. Also important to theconcept is the identification of selective steps in these chemicalsynthesis processes where one or more of the reactants has either a netpositive or negative charge, or to create such suitable reagents forthese processes.

One method for combinatorial oligonucleotide synthesis is shown in FIG.14. This method begins with a set of selectively addressablemicro-locations (140) whose surfaces have been derivatized with blockedprimary amine (X—NH—) groups (142). The initial step in the processinvolves selective deblocking of electrodes using a charged deblockingreagent (144). In this case, the reagent would carry a positive (+)charge. The process is carried out by applying a negative potential tothose electrodes being de-blocked, and a positive potential to thosewhich are to remain protected (FIG. 14(B)). Application of positive andnegative potentials to selective electrodes causes the charged reagentsto be concentrated at those micro-locations being de-blocked, andexcludes the reagents from the other electrode surfaces.

In the second step, chemical coupling of the first base, in this casecytosine, to the deblocked micro-locations is carried out by simplyexposing the system to the phosphoramidite reagent (x-C) (146). The (C)nucleotide couples to de-blocked micro-location surfaces, but not to anyof the blocked electrode surfaces (FIG. 14(C) and (D)). At this pointnormal phosphoramide chemistry IS carried out until the next de-blockingstep.

At the second de-blocking step (FIG. 14(D)), those electrode positionswhich are to be coupled with the next base are made negative, and thosewhich are to remain protected are made positive. The system is nowexposed to the next base to be coupled, in this case (x-A) (148), andselective coupling to the de-blocked micro-location is achieved (FIGS.14(E) and (F)). The coupling and deblocking procedures are repeated,until all the different DNA sequences have been synthesized on each ofthe addressable micro-location surfaces.

The above example represents one possible approach for the synthesis ofnucleic acids. Another approach involves a complete water soluble DNAsynthesis. In this case, charged water soluble coupling agents, such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCA), is used to carryout oligonucleotide synthesis with water soluble nucleotide derivatives.This approach would have significant advantages over present organicsolvent based methods which require extensive blocking of the basemoieties. Water soluble synthesis would be less expensive and eliminatethe use of many toxic substances used in the present organic solventbased processes. A third approach involves the use of charged monomers.

In addition to DNA synthesis, a similar process can be developed forpeptide synthesis, and other complex polymers. Examples contemplated inthis disclosure represent the initial potential for this technique, andare based on organic solvent based synthetic procedures for DNA orpeptide synthesis.

The recipes for buffers, solutions, and media in the following examplesare described in J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecularcloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

EXAMPLE 1 Oligomer Reagents

Synthetic DNA probes were made using conventional phosphoramiditechemistry on Applied Biosystems automated DNA synthesizers. Oligomerswere designed to contain either a 5′ amino or a 3′ ribonucleosideterminus. The 5′ functionality was incorporated by using the ABIAminolink 2 reagent and the 3′ functionality was introduced byinitiating synthesis from an RNA CPG support. The 3′ ribonucleotideterminus can be converted to a terminal dialdehyde by the periodateoxidation method which can react with primary amines to form a Schiff'sbase. Reaction conditions were as follows: Dissolve 20-30 O.D. oligomerin water to a final concentration of 1 OD/μl. Add 1 vol of 0.1M sodiumacetate, pH 5.2 and 1 vol 0.45M sodium periodate (made fresh in water).Stir and incubate reaction for at least 2 hours at ambient temperature,in the dark. Load reaction mix onto a Sephadex G-10 column (pasteurpipette, 0.6×5.5 cm) equilibrated in 0.1M sodium phosphate, pH 7.4.Collect 200 μl fractions, spot 2 μl aliquots on thin layerchromatography (TLC) and pool ultra violet (UV) absorbing fractions.

The following oligomers contain 3′ ribonucleoside termini (U):

(Sequence ID No. 1) ET12R 5′- GCT AGC CCC TGC TCA TGA GTC TCU(Sequence ID No. 2) CP-1 5′- AAA AAA AAA AAA AAA AAA AAU(Sequence ID No. 3) AT-A15′- CTA CGT GGA CCT GGA GAG GAA GGA GAC TGC CTG U (Sequence ID No. 4)AT-A2 5′- GAG TTC AGC AAA TTT GGA GU (Sequence ID No. 5) AT-A35′- CGT AGA ACT CCT CAT CTC CU (Sequence ID No. 6) AT-A45′- GTC TCC TTC CTC TCC AGU (Sequence ID No. 7) AT-A55′- GAT GAG CAG TTC TAC GTG GU (Sequence ID No. 8) AT-A65′- CTG GAG AAG AAG GAG ACU (Sequence ID No. 9) AT-A75′- TTC CAC AGA CTT AGA TTT GAC U (Sequence ID No. 10) AT-A85′- TTC CGC AGA TTT AGA AGA TU (Sequence ID No. 11) AT-A95′- TGT TTG CCT GTT CTC AGA CU (Sequence ID No. 12) AT-A105′- CAT CGC TGT GAC AAA ACA TU

Oligomers containing 5′ amine groups were generally reacted withfluorophores, such as Texas Red (TR, ex. 590 nm, em. 610 nm). Sulfonylchlorides are very reactive towards primary amines forming a stablesulfonamide linkage. Texas Red-DNA conjugates were made as follows:Texas Red sulfonyl chloride (Molecular Probes) was dissolved in dimethylformamide (DMF) to a final concentration of 50 mg/ml (80 mM). Oligomerwas dissolved in 0.4M sodium bicarbonate, pH 9.0-9.1, to a finalconcentration of 1 O.D./μl (5.4 mM for a 21-mer). In a micro test tube,10 μl oligomer and 20 μl Texas Red was combined. Let reaction proceed inthe dark for 1 hour. Quench reaction with ammonia or hydroxylamine,lyophilize sample and purify by PAGE (Sambrook et al., 1989, supra).

The following oligomers contain 5′ amino termini:

(Sequence ID No. 13) ET21A 5′- Aminolink2 - TGC GAG CTG CAG TCA GAC AT(Sequence ID No. 14) ET10AL 5′- Aminolink2 - GAG AGA CTC ATG AGC AGG(Sequence ID No. 15) ET11AL 5′- Aminolink2 - CCT GCT CAT GAG TCT CTC(Sequence ID No. 16) T2 5′- Aminolink2 - TTT TTT TTT TTT TTT TTT TT(Sequence ID No. 17) RC-A15′- Aminolink2 - CAG GCA GTC TCC TTC CTC TCC AGG TCC ACG TAG(Sequence ID No. 18) RC-A2 5′- Aminolink2 - CTC CAA ATT TGC TGA ACT C(Sequence ID No. 19) RC-A3 5′- Aminolink2 - GGA GAT GAG GAG TTC TAC G(Sequence ID No. 20) RC-A4 5′- Aminolink2 - CTG GAG AGG AAG GAG AC(Sequence ID No. 21) RC-A5 5′- Aminolink2 - CCA CGT AGA ACT GCT CAT C(Sequence ID No. 22) RC-A6 5′- Aminolink2 - GTC TCC TTC TTC TCC AG(Sequence ID No. 23) RC-A7 5′- Aminolink2 - GTC AAA TCT AAG TCT GTG GAA(Sequence ID No. 24) RC-A8 5′- Aminolink2 - ATC TTC TAA ATC TGC GGA A(Sequence ID No. 25) RC-A9 5′- Aminolink2 - GTC TGA GAA CAG GCA AAC A(Sequence ID No. 26) RC-A10 5′- Aminolink2 - ATG TTT TGT CAC AGC GAT G

EXAMPLE 2 Electronically Addressable Micro-locations on aMicrofabricated Device—Polylysine Method

Microelectrodes were fabricated from microcapillary tubes (0.2 mm×5 mm).The microcapillaries were filled with 18-26% polyacrylamide containing0.1-1.0% polylysine and allowed to polymerize. The excess capillary wasscored and removed to prevent air bubbles from being trapped within thetubes and to standardize the tube length. Capillaries were mounted in amanner such that they shared a common upper buffer reservoir and hadindividual lower buffer reservoirs. Each lower buffer reservoircontained a platinum wire electrode.

The top surface of the microcapillary in the upper reservoir wasconsidered to be the addressable micro-location. Upper and lowerreservoirs were filled with 0.1 M sodium phosphate, pH 7.4 and prerunfor 10′ at 0.05 mA constant using a BioRad 500/1000 power supply.Pipette 2 μl (0.1 O.D.) periodate oxidized ET12R into the upperreservoir while the power is on and electrophorese for 2-5 minutes atconstant current. Reverse polarity so that the test capillary is nowbiased negative and electrophorese an additional 2-5 minutes. UnboundDNA is repulsed while the covalently attached DNA remains.

Aspirate upper reservoir buffer and rinse with buffer. Disassembleapparatus and mount a fresh reference capillary. Refill reservoir andadd fluorescently labeled complement DNA, i.e., ET10AL-TR.Electrophoretically concentrate the oligomer at the positively biasedtest micro-location for 2-5 minutes at 0.05 mA constant. Reverse thepolarity and remove unbound complement. Remove test capillary andexamine by fluorescence. Negative control for nonspecific binding wasperformed as described above substituting a noncomplementary DNAsequence ET21A-TR for ET10AL-TR.

A cross-section of the capillary micro-locations were examined under aJena epifluorescent microscope fitted with a Hamamatsu ICCD cameraimaging system. Fluorescent results indicate that complement ET10AL-TRhybridized to the binding entity/capture sequence and remainedhybridized even when the potential was changed to negative. ET21A-TRnoncomplement was not retained at the test capillary when the potentialwas reversed.

EXAMPLE 3 Electronically Addressable Micro-locations on aMicrofabricated Device—Succinimidyl Acrylate Method

This example describes an alternative attachment chemistry whichcovalently binds the 5′ terminus of the oligomer. Capillaries werefabricated as described above except that 1% succinimidyl acrylate(Molecular Probes) was substitute for the polylysine. The capillarieswere made fresh because the succinimidyl ester reacts with primaryamines and is labile, especially above pH 8.0. The capillaries weremounted as described above and the reservoirs were filled with 0.1 Msodium phosphate, pH 7.4. Prerun the capillaries for 10 minutes at 0.05mA. Pipette 2 μl ET10AL (0.1 O.D.), which contains a 5′ amino terminus,into the upper reservoir while the power is on and electrophorese for2-5 minutes. Reverse polarity so that the test capillary is now biasednegative and electrophorese an additional 2-5 minutes. Unbound DNA isrepulsed while the covalently attached DNA remains.

Aspirate upper reservoir buffer and rinse with buffer. Unmount thereference capillary and mount a fresh reference capillary. Refillreservoir and add fluorescent labeled complement oligomer, ET11AL-TR andelectrophorese as described above. Negative control for nonspecificbinding was performed as described above substituting a noncomplementDNA sequence ET21A-TR for ET11AL-TR.

Fluorescent results indicate that complement ET11AL-TR hybridized to thecapture sequence and remained hybridized even when the potential waschanged to negative. ET21A-TR noncomplement was not retained at theworking capillary when the potential was reversed.

EXAMPLE 4 Electronically Controlled Fluorescent Dye DetectionProcess-PAGE

DNA dyes such as ethidium bromide (EB) become fluorescent whenintercalated into DNA. The fluorescence and binding affinity is greaterwhen the DNA is double stranded than single stranded. Preparecapillaries as in Example 1 and hybridize as described above. EB wasadded to the solution (˜0.05 mM EB final concentration) and the testcapillary was biased negative because EB is positively charged. Thecapillaries were observed by epifluorescence at 550 nm excitation and600+ nm emission. Both the hybridized and unhybridized micro-locationsshowed red fluorescence (from EB).

The capillaries were re-mounted biased positive to repulse EB. Maintainconstant current at 0.05 mA for 0.03 Volt-Hours.

Capture Target Normalized Signal ET10AL ET11AL (Pos.) >200 ET10AL ET21A(Neg.) 1

Fluorescence at the unhybridized micro-locations diminished while thehybridized capillary retained fluorescence. Fluorescent signal wasmeasured using an ICCD camera imaging system and represent peakfluorescent intensities. The signal to noise ratio would be >>1000 foldif the entire fluorescent signal area was integrated. This demonstratesa method for increasing signal to noise ratios and thus the dynamicrange of the assay.

EXAMPLE 5 Electronically Addressable Locations on Metal Substrates

Aluminum (Al) and gold (Au) wire (0.25 mm, Aldrich) was reacted with 10%3-aminopropyltriethoxysilane (APS) in toluene. The APS reagent reactsreadily with the oxide and/or hydroxyl groups on the metal surface toform covalent bonds between the oxide and/or hydroxyl groups and theprimary amine groups. No pretreatment of the aluminum was necessary. Thegold wire was subjected to electrolysis in 5×SSC solution to form anoxide layer. Alternatively the metal wire can be oxidized by aperchloric acid bath.

The APS reaction was performed as follows: Wires were cut to 3 inchesand placed in a glass dish. Toluene was added to completely cover thewires and the temperature was brought to 50-60° C. on a heat plate. APSwas added to a final concentration of 10%. Mix solution and continue thereaction for 30 minutes. Rinse 3 times with copious volumes of toluene,then rinse 3 times with copious volumes of alcohol and dry in 50° C.oven. The APS treated wire can then be reacted with an aldehyde to forma Schiff's base. Binding entity ET12R was periodate oxidized asdescribed elsewhere. The electrodes were placed in a reservoir ofdegassed water. Power was applied at 0.05 mA constant for about 30seconds. Activated ET12R was immediately added. Power was applied, theliquid was aspirated and fresh water was added and then aspirated again.The test (biased positive) and reference electrodes were placed inHybridization Buffer (HB, 5×SSC, 0.1% SDS) containing fluorescentlabeled complement DNA, ET10-TR. After 2 minutes the electrodes werewashed three times for one minute each in Wash Buffer (1×SSC, 0.1% SDS)and observed by fluorescence (ex. 590 nm, em. 610 nm).

Results demonstrate that ET12R was specifically coupled to the treatedmetal surfaces. The test electrode was fluorescent while the referenceelectrode was not. Nonspecific adsorption of the DNA to the metal wasprevented by the presence of SDS in the Hybridization Buffer. Attachmentto gold substrates by electrolysis and subsequent APS treatment waseffective. Signal obtained was significantly stronger than observed withnon-oxidized gold. More importantly, this example showed that the metalsurfaces could be chemically functionalized and derivatized with abinding entity and not become insulated from the solution. The APSmethod represents one of many available chemistries to form DNA-metalconjugates.

EXAMPLE 6 Electronically Controlled Fluorescent Dye DetectionProcess—Metal Wire

DNA-aluminum electrode substrates were prepared and hybridized asdescribed in Example 5. A hybridized and an unhybridized DNA-Alelectrode were processed with an underivatized Al wire as the reference.EB was added to the solution and the test DNA electrodes were biasednegative to attract the dye. The solution was aspirated and fresh bufferwas added. The metal surfaces were examined under the microscope.

Remount the device and apply a positive potential for a definedvolt-hour. The buffer was aspirated, the electrodes were observed byepifluorescence. This was repeated until there was a significantdifference in fluorescence between the hybridized and unhybridized metalsurfaces.

Capture Target Normalized Signal ET12R ET10AL (Pos.) >140 ET12R None(Neg.) 1

Fluorescence at the unhybridized metal surfaces diminished while thehybridized metal surfaces retained fluorescence. Fluorescent signal wasmeasured using an ICCD camera imaging system and represent peakfluorescent intensities. The signal to noise ratio would be >>1000 foldif the entire fluorescent signal area was integrated. This exampledemonstrates a method for increasing signal to noise ratios and thus thedynamic range of the assay. Similar results were obtained usingcapillary gel configuration, suggesting that electrochemical effects donot significantly affect the performance of the assay.

EXAMPLE 7 Active Programmable Electronic Matrix (APEX)—Micro-machineFabrication

A radial array of 6 addressable 250 μm capillary locations wasmicro-machined. The device has a common upper reservoir and separatelower reservoirs such that the potential at each micro-location isindividually addressable. A unique oligomer binding entity is localizedand coupled to a specific capillary micro-location by the methodsdescribed elsewhere. The test micro-location has a positive potentialwhile the other micro-locations have negative potentials to preventnonspecific interactions.

The array is washed and then hybridized with a complementaryfluorescently labeled DNA probe. The array is washed to remove excessprobe and then observed under an epifluorescent microscope. Only thespecifically addressed micro-location will be fluorescent. The processwill be repeated with another binding entity at another location andverified by hybridization with a probe labeled with another fluorescentmoiety.

DNA sequences are specifically located to predetermined positions withnegligible crosstalk with the other locations. This enables thefabrication of micromatrices with several to hundreds of uniquesequences at predetermined locales.

EXAMPLE 8 Active, Programmable Electronic Matrix(APEX)—Microlithographic Fabrication

An 8×8 matrix of 50 μm square aluminum electrode pads on a silicon wafer(see FIG. 3) was designed, fabricated and packaged with a switch box(see Device Fabrication Section for details). Several materials andprocess improvements, as described below, were made to increase theselectivity and effectiveness of the chip.

8a) Selection of Topcoat

The APS process involves the entire chip. Selectivity of thefunctionalization process was dependent on the reactivity of the chipsurfaces. In order to reduce functionalization and subsequent DNAattachment of the areas surrounding the micro-locations, a material thatis less reactive to APS than SiO₂ or metal oxide is needed. Photoresistsand silicon nitride were tried. The different topcoats were applied tosilicon dioxide chips. The chips were examined by epifluorescence andthe then treated with APS followed by covalent attachment of periodateoxidized polyA RNA sequences (Sigma, MW 100,000). The chips werehybridized with 200 nM solution of Texas Red labeled 20-mer (T2-TR) inHybridization Buffer, for 5 minutes at 37° C. The chips were washed 3times in WB and once in 1×SSC. The chips were examined by fluorescenceat 590 nm excitation and 610 nm emission.

Silicon nitride was chosen because it had much less reactivity to APSrelative to silicon dioxide and was not inherently fluorescent like thephotoresist tested. Other methods such as UV burnout of the backgroundareas are also possible.

8b) APEX Physical Characterization

A finished matrix chip was visually examined using a Probe Test Station(Micromanipulator Model 6000) fitted with a B & L microscope and a CCDcamera. The chip was tested for continuity between the test pads and theouter contact pads. This was done by contacting the pads with themanipulator probe tips which were connected to a multimeter. Continuityensures that the pads have been etched down to the metal surface. Thepads were then checked for stability in electrolytic environments. Themetal wires were rated to handle up to 1 mA under normal dry conditions.However, reaction to a wet environment was unknown. A drop (1-5 μl) ofbuffered solution (1×SSC) was pipetted onto the 8×8 matrix. Surfacetension keeps the liquid in place leaving the outer contact pad areadry. A probe tip was contacted to a contact pad and another probe tipwas contacted with the liquid. The current was incremented up to 50 nAat max voltage of 50 V using a HP 6625A power supply and HP3458A digitalmultimeter.

The initial fabrication consisted of the silicon substrate, a silicadioxide insulating layer, aluminum deposition and patterning, and asilicon nitride topcoat. These chips were not very stable in wetenvironments because the metal/nitride interface was physical in natureand electrolysis would undermine the nitride layer. This would result inthe pads being electrically shorted. Furthermore, silicon nitride and Alhave different expansion coefficients such that the nitride layer wouldcrack when current was applied. This would allow solution to contact themetal directly, again resulting in electrolysis which would furtherundermine the layer.

The second fabrication process included a silicon dioxide insulatinglayer between the aluminum metal and silicon nitride layers. Silicondioxide and Al have more compatible physical properties and form abetter chemical interface to provide a more stabile and robust chip.

8c) DNA Attachment

A matrix chip was functionalized with APS reagent as described inExample 5. The chip was then treated with periodate oxidized polyA RNA(Sigma, average MW 100,000). The chip was washed in WB to remove excessand unbound RNA. This process coated the entire chip with the capturesequence with a higher density at the exposed metal surfaces than at thenitride covered areas. The chip was hybridized with a 200 nM solution ofT2-TR in HB for 5 minutes at 37° C. Then washed 3 times in WB and oncein 1×SSC for one minute each at ambient temperature. The chip wasexamined by fluorescence at 590 nm excitation and 610 nm emission.

The opened metal areas were brightly fluorescent and had the shape ofthe pads. Low fluorescent intensities and/or irregular borders wouldsuggest that the pads were not completely opened. Additional plasma etchtimes would be recommended.

8d) Electronically Controlled Hybridization

Active hybridization was performed by using a chip from Example 8c andbiasing one micro-location positive. This was done by using the switchbox which would also automatically bias the remaining micro-locationsnegative or by using an external solution electrode. Three microlitersof water was deposited on the matrix pads only. A current, ˜1-5 nA, wasapplied for several seconds and 0.1 pmole of T2-TR was added to thesolution. The liquid was removed and the chip was dried and examined byfluorescence at Texas Red wavelengths (ex.590 nm, em.610 nm).

Only the positively biased micro-location was fluorescent. This can berepeated many times to hybridize other micro-locations selectively.Additionally, the fluorescence DNA at one micro-location can betranslocated to another micro-location by biasing the initial locationnegative and the destination positive.

8e) Electronically Controlled Addressing and Device Fabrication

The matrix was functionalized with APS as described above. Bindingentity CP-1 was activated by periodate oxidation method. Fourmicro-locations were biased positive in the matrix and the remainderwere biased negative. Two microliters of water was deposited on thematrix and a current was applied. Binding entity, CP-1, was added andallowed to concentrate at the designated locations. The liquid wasremoved, the chip was rinsed briefly with water and two microliters ofwater was deposited on the chip. Again, current was applied for severalseconds and 0.1 pmole of T2-TR was added. The liquid was removed after ashort time and the entire chip was washed in WB, 3 times. The chip wasdried and examined for fluorescence.

Results indicate that the positively biased micro-locations werefluorescent. This example demonstrates the selective addressing ofmicro-locations with a specific binding entity, the localization andcovalent coupling of sequences to the micro-locations, and the specifichybridization of complementary target sequences to the derivatizedmicro-locations.

8f) Genetic Typing APEX Chip

DNA binding entities with 3′ ribonucleoside termini are synthesizedwhich are specific for the polymorphisms of HLA gene dQa. The bindingentities are activated by periodate oxidation as described above. Thereverse complements are also synthesized with 5′ amino termini and areconjugated with fluorophores, such as Texas Red, Rhodamine or Bodipydyes, as described elsewhere. The micro-locations are functionalizedwith primary amines by treatment with APS, as described elsewhere. Acouple microliters of solution are placed over the matrix but leavingthe contact pads dry. A specific micro-location is addressed by biasingthat micro-location positive, the periodate oxidized DNA oligomer isadded, ˜0.1 pmole, and is translocated and covalently coupled to thatlocation. The polarity is reversed and the unbound binding entitymolecules are removed. This is repeated for another binding entity atanother addressed micro-location until all the unique binding entitiesare bound to the chip. The chip is then hybridized to individualfluorescently labeled complement sequences to determine the specificityof the coupling reaction as well as en masse to visualize all addressedmicro-locations at once. On the same chip which is denatured to removecomplementary oligomers (10 minutes at 90° C. in 0.05% SDS), theaddressed micro-locations are hybridized with unlabeled reversecomplements or genomic DNA. Detection is via the fluorescent dyedetection assay as described elsewhere.

Results will demonstrate that micro-locations are specifically addressedwith unique binding entities. Nonspecific binding to negatively biasedmicro-locations will be negligible. The device and associated bindingentity chemistry is stable under denaturation conditions, thus makingthe addressed and fabricated device reusable. Alternative methods fordenaturing the hybrids would be to increase the current and/or increasethe time it is applied.

EXAMPLE 9 Electronic Stringency Control

The ability of the device to affect electronic stringency control isdemonstrated with the Ras oncogene model system. A single base pairmismatch adversely affects the melting temperature (Tm), a measure ofthe stability of the duplex. Traditional methods to discriminate betweenmismatch and perfect match (i.e., stringency control) rely ontemperature and salt conditions. Stringency can also be affected by theelectrophoretic potential. Oligomers listed below can be paired suchthat resulting hybrids have 0-2 mismatches. Oligomer binding entitiesare coupled to the micro-location and hybridized as described elsewhere.The polarity at the micro-location is then reversed and the hybrids aresubjected to constant current for a given time, or defined power levelsto denature the mismatch without affecting the perfect match.

(Sequence ID No. 27) Ras-G5′- GGT GGT GGG CGC CGG CGG TGT GGG CAA GAU -3′ (Sequence ID No. 28)Ras-1 3′- CC GCG GCC GCC ACA C - Aminolink2 -5′ (Sequence ID No. 29)Ras-2 3′- CC GCG GCA GCC ACA C - Aminolink2 -5′ (Sequence ID No. 30)Ras-3 3′- CC GTG GCA GCC ACA C - Aminolink2 -5′ (Sequence ID No. 31)Ras-T 5′- GGT GGT GGG CGC CGT CGG TGT GGG CAA GAU -3′

Microelectrodes are fabricated from microcapillary tubes as describedelsewhere. Binding entities Ras-G is periodate oxidized and covalentlybound to the addressed micro-location. Ras-G micro-location is thenhybridized with Ras-1-TR which is the perfect match, Ras-2-TR which is aone base pair mismatch (G-A) or Ras-3-TR which is a two base pairmismatch (G-A and G-T). The micro-locations are examined fluorescentlyto verify whether complementary sequences are hybridized and to whatextent. The microcapillaries are re-mounted and subjected to controlledtime at constant current until the mismatched hybrids are removedwithout significantly affecting the perfectly matched hybrids.

Results will indicate that stringency could be affected by theelectrophoretic potential. This example demonstrates that eachmicro-location can have individual stringency control, thus overcomes amajor obstacle to large scale parallel processing techniques which hadbeen limited to a single common stringency level.

1. A self-addressable, microelectronic device adapted to receive asolution including solvent molecules, comprising: a substrate comprisingat least one addressable microscopic location, each location comprisinga selectively addressable functioning DC mode microelectrode supportedby the substrate, wherein 5% to 25% of the surface of saidmicroelectrode is accessible to said solvent molecules; a permeationlayer disposed adjacent to the selectively addressable microelectrode; acurrent source operatively connected to the selectively addressablemicroelectrode; and an attachment layer adjacent to the permeation layerfor the covalent coupling of specific binding entities, said attachmentlayer having 10⁵-10⁷ functionalized locations/μm².
 2. Themicroelectronic device of claim 1 wherein the attachment layer isdisposed upon the permeation layer.
 3. The microelectronic device ofclaim 1, wherein the substrate includes a base and an overlyinginsulator.
 4. The microelectronic device of claim 3, wherein the base issilicon, glass, silicon dioxide, plastic, or a ceramic material.
 5. Themicroelectronic device of claim 3, wherein the base material is silicon,6. The microelectronic device of claim 1, wherein the substrate issilicon, glass, silicon dioxide, plastic, or a ceramic material.
 7. Themicroelectronic device of claim 1, wherein each of said at least oneaddressable microscopic location further comprises a metal oxide layeradjacent to said permeation layer thereby providing a base for thecovalent binding of said permeation layer.
 8. The microelectronic deviceof claim 1, wherein said permeation layer is formed fromaminopropyltriethoxy silane.
 9. The microelectronic device of claim 8,wherein said permeation layer and said attachment layer form a combinedlayer formed from aminopropyltriethoxy silane.
 10. The microelectronicdevice of claim 1, wherein said substrate comprises multiple addressablemicroscopic locations.